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The TREX1 3' Exonuclease and Autoimmune Disease: Structural and Biochemical Analysis of Disease Mutants Involved in Autoimmune Dysfunction

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abstract
The homodimeric TREX1 protein is a member of the DnaQ family of exonucleases and catalyzes the major 3′ exonuclease activity detected in mammalian cell extracts. Mutations within the Trex1 gene are the underlying cause of multiple autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus (SLE), and retinal vasculopathy and cerebral leukodystrophy (RVCL). Several cellular roles have been proposed for TREX1 including degradation of nicked genomic DNA during the granzyme A cell death pathway, disposing of single-stranded DNA aberrant replication intermediates, and degrading DNA derived from endogenous retroelements. Four conserved acidic residues (D18, E20, D130, D200) coordinate two divalent cations within the TREX1 active site. Metal ion A directs nucleophilic attack on the scissile phosphate and metal ion B stabilizes the pentacovalent intermediate. A conserved histidine residue in the TREX1 active site activates a water molecule coordinated to metal A to initiate catalysis. Specific mutations within the vicinity of the TREX1 active site have been linked to dominant AGS (D200H, D200N), recessive AGS (V201D), and FCL (D18N). Recent evidence suggests that dominant TREX1 active site mutants are defective in their double-stranded DNA degradation activity. This work addresses several important questions pertaining to TREX1 catalysis and how TREX1 mutation within the active site may lead to autoimmunity. What role does the binding of metal and DNA play in arranging the TREX1 active site for catalysis? Are there global or local structural changes in the TREX1 protein resulting from mutation that might inhibit TREX1 cellular function? Does the context of the DNA 3' terminus affect mutant TREX1 exonucleolytic activity? Can the dominant TREX1 mutants inhibit wild type TREX1 activity on both single- and double-stranded DNA substrates? The X-ray crystal structures of the wt apoprotein as well as TREX1 active site mutants were determined in order to identify structural changes that may be important in TREX1-mediated autoimmunity. The catalytic histidine likely acts as a switch between an active and a resting state for a TREX1 protomer. The apparent loss of mobility in this residue observed in the TREX1 mutant structures may contribute to diminished catalytic activity. Mutation within the active site vicinity alters (D18N and V201D) or precludes (D200H and D200N) the coordination of metal ion A and likely contributes to the observed decreased catalytic function of the mutant enzymes. The activities of the TREX1 active site mutants were determined on model substrates to evaluate the possible contributions of different proposed TREX1 substrates to the development of an aberrant immune response. The dominant active site mutants are inactive on dsDNA regardless of the context of the 3' terminus. Furthermore, these dominant mutants are able to inhibit wt TREX1 activity on double-, but not single-stranded DNA substrates by competitively binding and protecting the DNA 3' termini. The accumulation of double-stranded DNA in the cell due to a loss of TREX1 function may result in the aberrant recognition of self DNA that could stimulate an autoimmune response.
subject
TREX1
3' DNA Exonuclease
Autoimmune Disease
contributor
Bailey, Suzanna L (author)
Alexander, Rebecca (committee chair)
Hollis, Thomas (committee member)
Horita, David A (committee member)
Perrino, Fred W (committee member)
Poole, Leslie B (committee member)
date
2010-05-05T20:39:55Z (accessioned)
2010-06-18T18:57:42Z (accessioned)
2010-05-05T20:39:55Z (available)
2010-06-18T18:57:42Z (available)
2010-05-05T20:39:55Z (issued)
degree
Biochemistry & Molecular Biology (discipline)
identifier
http://hdl.handle.net/10339/14707 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work for access only to the Wake Forest University system for one year from the date below. After one year, release the entire work for access worldwide. (accessRights)
title
The TREX1 3' Exonuclease and Autoimmune Disease: Structural and Biochemical Analysis of Disease Mutants Involved in Autoimmune Dysfunction
type
Dissertation

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